Multiple array format

ABSTRACT

A multiple array substrate having multiple assay chambers, each assay chamber containing one or more arrays. The assay chambers include a form-in-place gasket between a substrate and a cover. Methods of forming such multiple array substrates are described. The assay chambers may further be associated with an analysis site for analyzing a sample solution contained within the assay chamber. Each assay chamber is in fluid communication with a port, and the ports are positioned in a spatial format adapted to interface to standard laboratory equipment for handling multiple fluids in parallel.

CROSS REFERENCE TO RELATED APPLICATION DESCRIPTION

1. Field of the Invention

The invention relates generally to manufacture of biochemical assays.More specifically, this invention relates to formation of fluid-tightseals, and containment structures relating to biological assays.

2. Background of the Invention

Biomolecular arrays (such as DNA or RNA arrays) are known and are used,for example, as diagnostic or screening tools. Such arrays includeregions of usually different sequence biomolecules (such aspolynucleotides or polypeptides) arranged in a predeterminedconfiguration on a substrate. These regions (sometimes referenced as“array features”) are positioned at respective locations (“addresses”)on the substrate. Biomolecular arrays typically are fabricated on planarsubstrates either by depositing previously obtained biomolecules ontothe substrate in a site specific fashion or by site specific in situsynthesis of the biomolecules upon the substrate. The arrays, whenexposed to a sample, will undergo a binding reaction with the sample andexhibit an observed binding pattern. This binding pattern can bedetected upon interrogating the array. For example all biomoleculetargets (for example, DNA) in the sample can be labeled with a suitablelabel (such as a fluorescent compound), and the label then can beaccurately observed (such as by observing the fluorescence pattern) onthe array after exposure of the array to the sample. Assuming that thedifferent biomolecule targets were correctly deposited in accordancewith the predetermined configuration, then the observed binding patternwill be indicative of the presence and/or concentration of one or morecomponents of the sample.

In use, the surface of the array is contacted with a solution containingthe sample. The speed and specificity of the binding reaction isdependent on several factors, including composition of the solution(ionic strength, pH, polarity, concentration and identity of thesample), temperature, and speed of mixing of the sample. Samples tend tobe expensive, precious, or limited to very small quantities. Therefore,current methods seek to reduce the amount of sample required by reducingthe amount of sample solution needed to contact the array. One currentmethod accomplishes this by confining the solution under a coverslipplaced on top of the array, creating a thin layer of solution betweenthe array surface and the coverslip. While this technique minimizes thequantity of solution required to contact the array, it eliminates theability to mix or stir the solution while the array is being exposed tothe solution. Mixing is thus limited to diffusion of the samplemolecules within the thin layer of solution between the coverslip andthe array surface. This results in very long incubation time, typically,over night and up to 24 hours. The coverslip method also does not allowone to seal the system (undesirable, because it allows evaporation atthe edges to occur). The coverslip method frequently results inspatially non-uniform binding because of variations in the flatness ofthe glass, the bending of the glass, and the thickness of the thin layerof solution. The coverslip method is also messy and clumsy to use;during the disassembly process, it is easy to scratch the array sincethe glass cover is in close contact to the array substrate.

As an alternative, some array manufacturers have created packages fortheir arrays. In one type of package, the array substrate is glued inplace and the package has a sealed inlet and outlet for the liquidsample. These packages usually have a relatively large (compared to thecoverslip systems) distance between the array surface and the matingopposite surface used to seal the chamber. This allows the samplesolution to flow across the array when injected into the package. Thepackage usually has to be oriented so that the array surface is verticalto allow the leading air bubble to float to the top and out. The samplevolumes in these packages are much larger than the coverslip method,typically greater than 100 microliters and up to 500 microliters ormore.

Another technique to create an assay chamber for an array is to place agasket between the array surface and a mating opposite surface and clampwith an external force. The distance between the two surfaces istypically between 0.5 mm and 1.0 mm. This distance is required to allowthe sample solution to flow in the chamber without being restricted bycapillary forces. An array enclosed in a package having an assay chamberis easier to handle and less likely to be damaged during use because themating surface is kept at a distance from the array surface. Mixing ofthe sample solution across the array surface is possible in the assaychamber by either pumping the liquid sample back and forth across thearray or rotating the package to move the liquid position within thesealed chamber. The problem with these types of chambers is the largevolume of liquid sample required to fill the volume between the twosurfaces while covering the array area. Large sample volumes aresometimes not possible or require dilution of the sample to fill thevolume. Dilution of the sample reduces sensitivity of the measurementand may extend the incubation time.

Ideally, one would like to approach the small volumes of the coverslipmethod while allowing for a more protected sealed system. One suchsystem is described in U.S. Pat. No. 6,361,486 to Gordon and U.S. Pat.No. 6,309,875 to Gordon. This technique uses variable orientationcentrifugation to move the sample in a thin cross section between thearray surface and the back plate. This technique uses centrifugation ofthe assay chamber to overcome capillary forces that deter mixing of thesample solution. By changing the orientation of the array during thecentrifugation, the sample is moved across the array and allowed to mixduring incubation. This system requires a reliable seal between thearray surface and the back plate that is sufficiently thin to allowsmall volumes of sample to cover large areas of the array.

To form a good seal, typically a compliant material is compressedbetween the two surfaces. It is difficult to find compliant materialthat is sufficiently thin and compatible with the chemistry used forthese biochemical experiments. Normal rubber sheet material is muchthicker than what is required for this application. To reduce the volumeof sample, a gasket thickness of 0.001″ to 0.003″ is required. Sheetmaterials typically become too flimsy or are relatively difficult tomanipulate at such a small scale. One available material is thin sheets(down to 0.002″) of silicone rubber. This material can be cut into thedesired shape and placed on the array surface. A back plate is thencarefully set in place, and pressure is applied to seal the assaychamber. In practice, this works, but the gasket is delicate, difficultto handle, and hard to keep in place while assembling the apparatus.Adhesives can be applied to one side of the silicone sheet. This allowsthe thin sheet of silicone rubber to be applied to the back plate andcut to the desired shape. Unwanted areas of the sheet are then peeledaway. The sheet of silicone rubber can also be die cut to form thegasket before it is applied to the back plate. This is a difficultprocess. The adhesive adds to the thickness of the gasket and has to becompatible with all the chemicals that might be used in the biochemicalassay. The silicone sheet material that forms the gasket must be wideenough (on the order of 1+ millimeters) to provide strength andstructural integrity to survive the process of applying the material tothe plate. Therefore, while creating a chamber on the order of about0.002″ thick is possible using thin silicone sheet material, it isinconvenient and results in relatively wide strips of sheet material onthe surface of the back plate (or, alternatively, the surface of thesubstrate).

There is thus a need for an array system allowing the use of relativelysmall amounts of sample solution while allowing the sample solution tobe mixed or moved across the surface of the array to speed the bindingreaction. Such an array system needs to have an assay chamber that isfluid tight to allow the sample solution to flow across the surface ofthe array and to be mixed without leaking.

SUMMARY OF THE INVENTION

The invention is thus addressed to the aforementioned deficiencies inthe art, and provides novel methods for making a fluid-tight seal aroundan array to provide an assay chamber for containing the sample solutionduring the binding reaction.

More generally, the invention provides a form-in-place gasket on agasket surface on a substrate as well as a method of making theform-in-place gasket on the gasket surface. The form-in-place gasketcomprises a suitable gasket material that is deposited onto the gasketsurface at the site where the finished gasket is desired, typicallyadjacent to an analysis site, e.g. site of a biochemical assay. Forembodiments in which the desired gasket is relatively thin, the gasketmaterial is selected to be a self-leveling, low viscosity, fluidmaterial that is essentially inert to the conditions under which theanalysis (such as a biochemical assay) is conducted. The method ofmaking the form-in-place gasket includes depositing the gasket materialin a predetermined configuration at the desired site on the gasketsurface, and then curing the gasket material to form the form-in-placegasket. A cover having a mating surface that is complementary to thegasket surface can be disposed against the gasket, forming a fluid tightseal. With the cover in place, the substrate, the cover, and theform-in-place gasket define an assay chamber, typically associated withthe analysis site.

The invention provides for a multiple array substrate havingform-in-place gaskets defining assay chambers around one or more arrays.Each assay chamber is in fluid communication with a port, and the portsare positioned in a spatial format adapted to interface to standardlaboratory equipment for handling multiple fluids in parallel.Particular embodiments have either eight or twelve assay chambers, eachin fluid communication with a port, the ports linearly positioned on 9mm centers. Other embodiments have either sixteen or twenty-four assaychambers, each in fluid communication with a port, the ports linearlypositioned on 4.5 mm centers. Other embodiments have either thirty-twoor forty-eight assay chambers, each in fluid communication with a port,the ports linearly positioned on 2.25 mm centers. Such a configurationallows for automated handling of processing of arrays, includingcontacting arrays with the solutions to be tested, washing buffers, etc.

Additional objects, advantages, and novel features of this inventionshall be set forth in part in the descriptions and examples that followand in part will become apparent to those skilled in the art uponexamination of the following specifications or may be learned by thepractice of the invention. The objects and advantages of the inventionmay be realized and attained by means of the instruments, combinations,compositions and methods particularly pointed out in the appendedclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will be understood from thedescription of representative embodiments of the method herein and thedisclosure of illustrative apparatus for carrying out the method, takentogether with the Figures, wherein

FIG. 1 is a flow chart describing a method according to the presentinvention.

FIG. 2A illustrates a substrate, a form-in-place gasket, and a cover;FIG. 2B illustrates form-in-place gasket including structural featuresdisposed on a substrate surface; FIG. 2C shows a form-in-place gasketforming a vent; FIG. 2D shows structural features including a vent, orport.

FIG. 3 is a cross-sectional view showing the profile of a bead of gasketmaterial on a substrate.

FIG. 4 shows a multiple array substrate with gaskets around individualarrays.

FIG. 5 is a drawing of a multiple array substrate formatted to interfacewith parallel fluid handling equipment.

FIG. 6 is a drawing of a multiple array substrate formatted to interfacewith parallel fluid handling equipment, wherein the multiple arraysubstrate provides for assay chambers arranged in a plurality of ranks.

FIG. 7 is a drawing of a multiple array substrate formatted to interfacewith parallel fluid handling equipment, wherein the multiple arraysubstrate provides for assay chambers arranged in a plurality of ranks.

FIG. 8 illustrates a form-in-place gasket on a cover forming a wellsuitable for holding an aliquot of sample fluid, with the arraysubstrate ready to be positioned on the form-in-place gasket.

To facilitate understanding, identical reference numerals have beenused, where practical, to designate corresponding elements that arecommon to the Figures. Figure components are not drawn to scale.

DETAILED DESCRIPTION

Before the invention is described in detail, it is to be understood thatunless otherwise indicated this invention is not limited to particularmaterials, reagents, reaction materials, manufacturing processes, or thelike, as such may vary. It is also to be understood that the terminologyused herein is for purposes of describing particular embodiments only,and is not intended to be limiting. It is also possible in the presentinvention that steps may be executed in different sequence where this islogically possible. However, the sequence described below is preferred.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “an insoluble support” includes a plurality of insolublesupports. In this specification and in the claims that follow, referencewill be made to a number of terms that shall be defined to have thefollowing meanings unless a contrary intention is apparent:

A form-in-place gasket, as the term is used herein, refers to a gasketwhich is formed on a gasket surface in a process that involvesdepositing a gasket material onto the gasket surface. The term“form-in-place gasket” also encompasses a plurality of discontinuousportions of gasket disposed on a surface, such as when formed bydepositing gasket material on the surface discontinuously and thencuring the gasket material. The gasket surface is the surface upon whichthe gasket is formed (or is intended to be formed). The mating surfaceis the surface that is complementary to the gasket surface and isdisposed against the gasket formed on the gasket surface (or is intendedto be disposed against the gasket formed on the gasket surface). Gasketmaterial references a fluid material having properties that render thefluid material suitable for formation of a gasket. As used below,“gasket” typically references a form-in-place gasket according to thepresent invention, unless the context clearly indicates otherwise.“Fluid tight” when used to describe a seal, a chamber, or other featurereferences an ability to resist flow of a fluid past an intendedboundary (typically defined by a gasket), but yet permits fluid flowwithin intended boundaries, such as on one side of a seal, into or outof a chamber via a port, or along the length of a channel. “Mixingfeature” references structures formed on a surface (e.g. by depositinggasket material on the surface) that, due to the geometry or physicalconfiguration of the structure, serves to aid mixing of the contents ofa chamber. In certain embodiments, a “substrate” may include materialsthat are homogenous, heterogenous, or otherwise, and may includeindividual component parts that are combined to produce the substrate.Similarly, in certain embodiments, a “cover” may include materials thatare homogenous, heterogenous, or otherwise, and may include individualcomponent parts that are combined to produce the cover. “Substantiallydefined”, as it relates to a substrate, a cover, and gasket“substantially defining” an assay chamber, means that the chamber neednot be totally enclosed (e.g. the chamber may have one or more ports, ororifices), and/or that other elements (other than the substrate, cover,and gasket) may define a portion (e.g. less than about 20% of thesurface area defining the assay chamber) of the assay chamber or maycontribute (e.g. up to about 20% of the surface area defining the assaychamber) to defining the assay chamber. “Substantially” in othercontexts means generally at least about 80% of the property or statereferred to, unless the context clearly dictates otherwise. “Pliable”references a property of a material which is pliant or compressible.“Self-leveling” references a property of a material which tends to havea certain given thickness under a given set of conditions, and inparticular references the property of certain gasket materials to flow,or to “slump”, (after being deposited on a gasket surface but prior tocompletion of curing) to a certain thickness, where the thicknessdepends on properties of the gasket material applied and the conditionsof application, including the conditions used for curing the gasketmaterial and the properties of the surface on which the gasket materialis deposited. “Non-slumping” references a property of a material whichdoes not flow, or which maintains an essentially constant conformation,after being deposited on a gasket surface but prior to completion ofcuring. Of course, non-slumping materials may be manipulated to resultin a changed conformation after being deposited on a gasket surface butprior to completion of curing, e.g. by being squeezed between asubstrate and a cover, and this does not alter their “non-slumping”property. “Uniform thickness” describes gaskets or gasket materialsapplied to a surface such that substantially the entire gasket orapplied gasket material has a given thickness (plus or minus about 20%),wherein the thickness of the gasket measured at various points varies byless than 20% of the given thickness of the gasket.

As used herein, polynucleotides include single or multiple strandedconfigurations, where one or more of the strands may or may not becompletely aligned with another. The terms “polynucleotide” and“oligonucleotide” shall be generic to polydeoxynucleotides (containing2-deoxy-D-ribose), to polyribonucleotides (containing D-ribose), to anyother type of polynucleotide which is an N-glycoside of a purine orpyrimidine base, and to other polymers in which the conventionalbackbone has been replaced with a non-naturally occurring or syntheticbackbone or in which one or more of the conventional bases has beenreplaced with a non-naturally occurring or synthetic base.

A “nucleotide” refers to a sub-unit of a nucleic acid (whether DNA orRNA or analogue thereof) which includes a phosphate group, a sugar groupand a nitrogen containing base, as well as analogs of such sub-units. A“nucleoside” references a nucleic acid subunit including a sugar groupand a nitrogen containing base. A “nucleoside moiety” refers to amolecule having a sugar group and a nitrogen containing base (as in anucleoside) as a portion of a larger molecule, such as in apolynucleotide, oligonucleotide, or nucleoside phosphoramidite. A“nucleotide monomer” refers to a molecule which is not incorporated in alarger oligo- or poly-nucleotide chain and which corresponds to a singlenucleotide sub-unit; nucleotide monomers may also have activating orprotecting groups, if such groups are necessary for the intended use ofthe nucleotide monomer. A “polynucleotide intermediate” references amolecule occurring between steps in chemical synthesis of apolynucleotide, where the polynucleotide intermediate is subjected tofurther reactions to get the intended final product, e.g. a phosphiteintermediate which is oxidized to a phosphate in a later step in thesynthesis, or a protected polynucleotide which is then deprotected. An“oligonucleotide” generally refers to a nucleotide multimer of about 2to 100 nucleotides in length, while a “polynucleotide” includes anucleotide multimer having any number of nucleotides. It will beappreciated that, as used herein, the terms “nucleoside” and“nucleotide” will include those moieties which contain not only thenaturally occurring purine and pyrimidine bases, e.g., adenine (A),thymine (T), cytosine (C), guanine (G), or uracil (U), but also modifiedpurine and pyrimidine bases and other heterocyclic bases which have beenmodified (these moieties are sometimes referred to herein, collectively,as “purine and pyrimidine bases and analogs thereof”). Suchmodifications include, e.g., methylated purines or pyrimidines, acylatedpurines or pyrimidines, and the like, or the addition of a protectinggroup such as acetyl, difluoroacetyl, trifluoroacetyl, isobutyryl,benzoyl, or the like. The purine or pyrimidine base may also be ananalog of the foregoing; suitable analogs will be known to those skilledin the art and are described in the pertinent texts and literature.Common analogs include, but are not limited to, 1-methyladenine,2-methyladenine, N6-methyladenine, N6-isopentyladenine,2-methylthio-N6-isopentyladenine, N,N-dimethyladenine, 8-bromoadenine,2-thiocytosine, 3-methylcytosine, 5-methylcytosine, 5-ethylcytosine,4-acetylcytosine, 1-methylguanine, 2-methylguanine, 7-methylguanine,2,2-dimethylguanine, 8-bromoguanine, 8-chloroguanine, 8-aminoguanine,8-methylguanine, 8-thioguanine, 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, 5-ethyluracil, 5-propyluracil,5-methoxyuracil, 5-hydroxymethyluracil, 5-(carboxyhydroxymethyl)uracil,5-(methylaminomethyl)uracil, 5-(carboxymethylaminomethyl)-uracil,2-thiouracil, 5-methyl-2-thiouracil, 5-(2-bromovinyl)uracil,uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester,pseudouracil, 1-methylpseudouracil, queosine, inosine, 1-methylinosine,hypoxanthine, xanthine, 2-aminopurine, 6-hydroxyaminopurine,6-thiopurine and 2,6-diaminopurine.

An “internucleotide bond” refers to a chemical linkage between twonucleoside moieties, such as a phosphodiester linkage in nucleic acidsfound in nature, or such as linkages well known from the art ofsynthesis of nucleic acids and nucleic acid analogues. Aninternucleotide bond may comprise a phospho or phosphite group, and mayinclude linkages where one or more oxygen atoms of the phospho orphosphite group are either modified with a substituent or replaced withanother atom, e.g. a sulfur atom, or the nitrogen atom of a mono- ordi-alkyl amino group. Such words as “bond,” “bound,” “binds,” or“binding,” may be used to express various modes of chemical binding,including covalent, ionic, hydrogen bonding, hydrophobic bonding, ormixed mode binding (combinations of the above); context may dictate whena specific meaning is permissible or required.

As used herein, the term “amino acid” is intended to include not onlythe L-, D- and nonchiral forms of naturally occurring amino acids(alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, valine), but also modified amino acids, amino acid analogs,and other chemical compounds which can be incorporated in conventionaloligopeptide synthesis, e.g., 4-nitrophenylalanine, isoglutamic acid,isoglutamine, ε-nicotinoyl-lysine, isonipecotic acid,tetrahydroisoquinoleic acid, α-aminoisobutyric acid, sarcosine,citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, β-alanine, 4-aminobutyric acid, and the like. An“oligopeptide” is a molecule containing from 2 to about 100 amino acidsubunits. “Polypeptide” refers to a molecule having any number of aminoacid subunits. “Biomolecule” refers to molecules generally derivablefrom living organisms, or analogues thereof. Biomolecules include, e.g.amino acids, oligopeptides, polypeptides, glycoproteins, nucleotidemonomers, oligonucleotides, polynucleotides, saccharides,polysaccharides, hormones, growth factors, peptidoglycans, or the like.The term “biomolecular fluid” refers to any fluid that comprisesbiological fluids, biomolecules, and/or other biological substances ormaterials. Some examples of biological fluids include blood, plasma,serum, solutions containing proteins or nucleic acids, urine, cerebralspinal fluid, saliva, enzymatic mixtures substances and other relatedsubstances and fluids that are well known in the analytical andbiomedical art.

The term “analysis site” refers to a location in a device where there isany use, singly or in combination, of chemical test reagents andmethods, electrical test circuits and methods, physical test componentsand methods, optical test components and methods, and biological testreagents and methods to yield information about an analyte, e.g. abiomolecular fluid or other substance to be analyzed. Such methods arewell known in the art and may be based on teachings of, e.g. TietzTextbook of Clinical Chemistry, 3d Ed., Sec. V, pp. 776-78 (Burtis &Ashwood, Eds., W. B. Saunders Company, Philadelphia, 1999); U.S. Pat.No. 5,997,817 to Chrismore et al. (Dec. 7, 1999); U.S. Pat. No.5,059,394 to Phillips et al. (Oct. 22, 1991); U.S. Pat. No. 5,001,054 toWagner et al. (Mar. 19, 1991); and U.S. Pat. No. 4,392,933 to Nakamuraet al. (Jul. 12, 1983), the teachings of which are hereby incorporatedby reference, as well as others. Analysis sites may include detectorsthat test electrochemical properties of the biomolecular fluid (e.g.conductivity), or they may include optical means for sensing opticalproperties of the biomolecular fluid (e.g. chemiluminescence,fluorescence, or dye activation vie enzymatic action), or they mayinclude biochemical reagents (e.g. antibodies, substrates, or enzymes)to sense properties (e.g. presence of antigens, clotting time, or clotlysis) of the biomolecular fluid. The analysis site may comprisebiosensing or reagent material that will react with an analyte (e.g.glucose) in the biomolecular fluid so that information about the analytemay be obtained. An analysis site may include one or more bioarrays or aportion of a bioarray. “Analysis component” references any reagent,circuit, component, detector, means, or the like mentioned in thisparagraph in relation to an analysis site, wherein the analysiscomponent is in operable relation to other elements of the analysissite, and may include biomolecules deposited in a bioarray on asubstrate. An analysis site may be, e.g. disposed adjacent to asubstrate or in operable relation to an array chamber or a portion of anarray chamber, and an analysis component may be in operable relation toa substrate.

An “array”, unless a contrary intention appears, includes any one, twoor three dimensional arrangement of addressable regions bearing aparticular chemical moiety or moieties (for example, polynucleotidesequences) associated with that region. A “bioarray” is an array ofbiomolecules. An array is “addressable” in that it has multiple regionsof different moieties (for example, different polynucleotide sequences)such that a region (a “array feature” or “spot” of the array) at aparticular predetermined location (an “address”) on the array willdetect a particular target or class of targets (although an arrayfeature may incidentally detect non-targets of that array feature). Inthe case of an array, the “target” will be referenced as a moiety in amobile phase (typically fluid), to be detected by probes (“targetprobes”) which are bound to the substrate at the various regions.However, either of the “target” or “target probes” may be the one whichis to be evaluated by the other (thus, either one could be an unknownmixture of polynucleotides to be evaluated by binding with the other).While probes and targets of the present invention will typically besingle-stranded, this is not essential. A “target solution” references amobile phase comprising the target. “Interrogating” the array refers toobtaining information from the array, especially information abouttargets binding to the array. An “array format” refers to one or morecharacteristics of the array, such as array feature position, arrayfeature size, or some indication of a moiety at a given location.“Hybridization assay” references a process of contacting a bioarray witha mobile phase containing target moieties. Further disclosure ofform-in-place gaskets, particularly as related to array formats may befound in three applications co-filed with this application, titled “Formin Place Gaskets for Assays” (Schleifer, docket 10010945), “FluidContainment Structure” (Schleifer, docket 10020753), “ImprovedHybridization Process for Bioarrays” (Schleifer and Ostrowski, docket10020292), each of which is hereby incorporated by reference in itsentirety.

The present invention provides a method of making a form-in-placegasket. Referring to FIG. 1, the method comprises first depositing asuitable gasket material onto a substrate 10 and then curing the gasketmaterial on the substrate 20 to produce the form-in-place gasket. Thegasket material is applied to the substrate in a predeterminedconfiguration to provide for a form-in-place gasket having a desiredconfiguration. The desired properties of the final form-in-place gasket,for example the spatial conformation of the gasket on the surface of thesubstrate, the desired dimensions and structural features of the gasket,and so on, will dictate the predetermined configuration. The gasketmaterial is applied to the gasket surface so as to result in the finalgasket having the desired configuration, for example to provide desiredstructural features such as conduits, chambers, mixing features, outletsand/or inlets when a cover is placed against the form-in-place gasket onthe substrate.

A fluid containment structure may be formed that includes a substratethat has a gasket surface with a form-in-place gasket disposed on thegasket surface. In the fluid containment structure, the form-in-placegasket is disposed around and marks the perimeter of an interior area onthe substrate. The interior area and the form-in-place gasket define awell that is adapted for retaining a fluid. The shape of the interiorarea may be altered depending on the desired use by altering theconfiguration of the form-in-place gasket. The fluid containmentstructure may be associated with or form a portion of an analysis sitewhere a sample fluid retained in the fluid containment structure may beanalyzed. The analysis site typically includes at least one analysiscomponent (e.g. an array of immobilized oligonucleotides) necessary forperforming, e.g. a biochemical assay, such as a binding reaction betweenan immobilized oligonucleotide and a complementary oligonucleotide inthe sample solution.

The gasket material may be deposited on a gasket surface in a variety ofpredetermined configurations, including a bead of gasket materialdeposited around the perimeter of a substrate to provide for afluid-tight seal when a cover is placed on the gasket. The desiredconfiguration may include, for example, a simple bead of gasket materialsurrounding the perimeter of an assay chamber, as well as more complexstructures. Referring now to FIG. 2, FIG. 2A shows a substrate 30 havinga gasket surface 32 with a form-in-place gasket 34 formed thereupon. Thegasket surface 32 is that part of the substrate surface 36 on which thegasket material is deposited. The form-in-place gasket 34 comprises asimple bead of cured gasket material around the perimeter of an interiorarea 38 of the substrate surface 36 defined by the form-in-place gasket34. A cover 40 having a mating surface 42 is adapted to be positioned inclose proximity to the substrate 30, as indicated by arrows 44. Themating surface 42 is that part of the cover 40 which lies adjacent theform-in-place gasket 34 when the cover 40 is disposed adjacent thesubstrate 30. The mating surface 42 is complementary to the gasketsurface 32 and is preferably smooth where it contacts the form-in-placegasket 34. The cover 40 is adapted to provide a tight seal by pressingthe form-in-place gasket 34 between the gasket surface 32 and the matingsurface 42. The gasket surface and the mating surface are eachpreferably planar, but in other embodiments may deviate from planar,e.g. portions of the gasket surface and mating surface may turn downwardor upward (i.e. in the direction of the arrows 44 or in the reversedirection), so long as the gasket surface and mating surface arecomplementary, or substantially parallel (meaning substantiallyequidistant from each other along their length) when the cover is inplace on the substrate, so that a tight seal may be formed. With thecover in place (in close proximity (“adjacent”) to the substrate), thesubstrate, the cover, and the form-in-place gasket define an assaychamber. In the absence of the cover, substrate 30 and form-in-placegasket 34 provide a fluid containment structure, as illustrated in FIG.2A.

The substrates shown in FIGS. 2A-2D are planar, but in alternateembodiments the substrate may have more complex structure, e.g.including one or more of recessed structures, elevated structures,channels, orifices, guides. For example, the interior area 38 of thesubstrate surface 36 defined by the form-in-place gasket 34 may have arecess in fluid communication with a channel formed in the interior area38 of the substrate surface 36, the channel leading through an orificeto an external fluid supply source, allowing, e.g. rinsing of theinterior surface during use, with a separate orifice serving as a drain.Such orifices may variously be referred to as ports, inlets, outlets,drains, vents, or such similar terms.

FIGS. 2B, 2C, and 2D provide a few examples of how the form-in-placegasket 34 may have a variety of structural features. FIG. 2B illustratesa form-in-place gasket 34 on a substrate 30 where the form-in-placegasket 34 has internal structural features 46. These structural features46 may serve functions such as enhancing mixing of fluids, partitioningfluids in separate reservoir chambers until needed for mixing orrinsing, or other desired functions that may be apparent to the skilledpractitioner given the disclosure herein. FIG. 2C illustrates aform-in-place gasket 34 with more internal structural features 46, thistime including an internal vent 48 that provides for enhanced fluid flowduring use of the illustrated device. FIG. 2D illustrates aform-in-place gasket 34 with a different structural feature 50 that mayfunction as an external vent or as a port to supply or remove fluid fromthe chamber. Some embodiments may provide for ports through thesubstrate 30 or through the cover 40 to supply or remove fluid from thechamber. In each of the embodiments illustrated in FIGS. 2B-2D, a cover40 may be disposed adjacent the substrate 30 with the gasket between thesubstrate and cover to form an assay chamber.

The gasket material may deposited on the substrate in predeterminedconfigurations which include structural features, such as, for example,gaps, protrusions, vents, channels, mazes, serpentine channels, bumps,sample inlets, and/or sample outlets. These structural features can beused to enhance performance of the system, such as by directing fluidflow, partitioning fluids, or enhancing mixing of fluids. Additionaldevices, such as chemically derivatized beads and filters, can be gluedin place and sealed as part of the gasket structure. Interior areas onthe substrate surface defined by form-in-place gasket may also serve aswells for retaining fluid (fluid containment structures). Multiple areasmay be defined on a single substrate (see FIG. 4), allowing differentsamples to be applied to and analyzed on a single substrate, thuspotentially reducing cost, increasing throughput, or increasing thenumber of different analytes which can be tested on a single substrate.It can be seen from the figures that the gasket material may bedeposited on the substrate to form continuous structures (those that maybe deposited without halting and restarting the application of thegasket material), like shown in FIG. 2A, or discontinuous structures(those that require halting the application of the gasket material andrestarting application at a different point on the substrate surface),like those shown in the other figures. In some embodiments, other fluidhandling features such as, for example, gaps, protrusions, vents,channels, mazes, serpentine channels, bumps, ports, sample inlets,and/or sample outlets may be present on the substrate or otherwiseassociated with the assay chamber.

The gasket material is selected to provide a form-in-place gasket havingsuitable thickness and flexibility to enable a fluid tight seal whereneeded for the desired configuration. In one embodiment, the gasketmaterial is cured on the substrate in the absence of a cover, with acover optionally being placed on the gasket after curing of the gasketmaterial. An alternate embodiment provides that the cover is put inplace before curing of the gasket material, thus providing aform-in-place gasket where the gasket is formed between the substrateand the cover. In one embodiment, the gasket material is selected sothat, when it is cured prior to positioning the cover over the assaysite, the cover may be removed from contact with the gasket withoutsignificant damage to the form-in-place gasket, allowing the cover (or adifferent cover) to be re-positioned over the assay site to form a seal(i.e. the gasket is re-usable). In an alternate embodiment, the cover isput in place prior to the curing of the gasket material, and then thegasket material is allowed to cure with the cover in place. This type ofseal typically leads to damage of the gasket upon removal of the cover;in this case the seal may be formed only once (i.e. gasket may not bere-used, or the seal may not be re-formed after breaking, or the sealnot intended to be broke in normal use of the device). This type of sealmay be termed a “non-releasable” seal, or a “permanent seal”, meaningthat the seal is not adapted to being broken and then reformed.

Any material having suitable characteristics may be used as a gasketmaterial. Gasket materials are generally fluid materials that can becured to provide a gasket having suitable characteristics. Selection ofa gasket material is determined relative to the intended application.Suitable gasket materials include, e.g. silicone sealants, urethanes,and polysulfides. Still other suitable gasket materials are, e.g. latex,and acrylic sealants. In all types of gaskets cured on a substrate inthe absence of a cover, a low durometer material is used to allow for acompression seal. Silicone sealant materials are available in manyformulations that are suitable for use in the process of makingform-in-place gaskets according to the current invention. For very thingaskets, with dimensions from about 20 to about 100 micrometers thick, aself-leveling, low viscosity, fluid material should be selected. Thickergaskets can use a wider range of materials including higher viscositymaterial to non-slumping or paste materials. For the relatively thingaskets, a suitable formulation should provide for a silicone gasketthat remains highly flexible and durable after curing. By using a lowviscosity (about 15,000 to about 50,000 cps, or centipoises) siliconethat is “self leveling”, a very small bead of silicone can be applied toa gasket surface. Being self-leveling, the small bead of silicone willspread out to a thin profile, or cross section. In some embodiments, thesilicone will have a viscosity in the range of about 20,000 to about40,000 cps, or even in the range of about 25,000 to about 35,000 cps. Inother embodiments, the viscosity may be in the range of about 50,000 toabout 80,000 cps. Other embodiments may use a gasket material that isnon-slumping. In certain embodiments of the invention, the preferredgasket material is a silicone sealant material such as RTV 118 availablefrom FE Silicones (Charlotte, N.C.), RTV 734 or 3-1753 (both availablefrom Dow Corning (Midland, Mich.)). An example of a paste siliconeadhesive that is a thermal cure silicone is GE 6124. Gasket materialsmay also be selected based on their hardening properties—a gasketmaterial that forms a soft, rounded profile gasket that is compressiblemay be desirable for forming fluid-tight seals where tolerances betweensubstrate and cover may vary; however, a less soft, less compressiblegasket may be desired for other applications.

The gasket material may be applied to the gasket surface by any suitablemethod, e.g. silk screen, brush, spray, or transfer process. Forexample, to apply a pattern of the gasket material using a pad transferprocess, a negative relief of the pattern is generated so that thedesired thickness of the adhesive is the depth of the relief in themold. The mold is then covered with the gasket material and pressed intothe mold, and the excess is scraped off. A flexible pad is then pressedonto the relief area and the gasket material is transferred from themold to the surface of the pad. The pad is then moved into the desiredposition for the gasket. As the pad contacts the surface (e.g. thesubstrate surface), again the gasket material is transferred from thepad onto the surface. A company that manufactures and distributes padprinting technologies is Printex, A Division Of Pemco Industries, Inc.(Poway, Calif.).

In one embodiment, the method of applying the gasket material to thegasket surface uses a dispensing system designed for adhesive sealants.The dispensing system has an x-y-z positioning system and isprogrammable to allow the application of a thin bead of silicone ontothe gasket surface in the desired configuration. A suitable system isthe Automove 403 and is available from Asymtek (Carlsbad, Calif.). Theuse of such a dispensing system is described below in the examples.

The gasket surface in certain embodiments should be relatively smooth.In an alternate embodiment, the gasket surface is etched, chemicallytreated, or scarified to provide greater adhesion of the gasketmaterial. After the gasket material is deposited in the predeterminedconfiguration at the desired site, the gasket material is allowed tocure to form the form-in-place gasket. Various methods of curing dependon the properties of the materials. One part adhesives have theadvantage of only needing to dispense, transfer, paint or spray onematerial and do not require any mixing of two or more materials prior touse or after in place. One part adhesives are cured depending propertiesof the material. For one part adhesives, curing can be done by moisturecure, such as moisture cure RTV silicone where moisture in the airreacts with the silicone. Typical cure times for these RTV silicones arefrom 1 to several days. In some embodiments the gasket material may beexposed to heat to cause or to speed up the curing process. Heat curegasket material, such as heat cure silicone, are cured by a process ofheating the material well above room temperature for a described time,typically 1 to 2 hours. There are also UV cure adhesives where thematerial is exposed to UV light. This are typically fast curing times inas little as 1 minute. Multiple part adhesives are also available wherea curing agent is mixed into the material before application, mixedduring the dispensing, or sprayed on before or after application. Thecuring agent is typically a catalyst to the curing process. Thedisadvantage of multipart adhesives is that one has to handle more thanone material and if premixed, a working time is associated with thematerial. A cover having a mating surface that is complementary to thegasket surface can be placed over the substrate, forming a fluid tightseal between the substrate and the cover.

The physical dimensions of the form-in-place gasket may be characterizedin terms of thickness, width, and length. Thickness is defined as theperpendicular distance from the gasket surface to most distal surface ofthe applied gasket, when the cover is not in place. When the cover is inplace, the thickness is the perpendicular distance between the gasketsurface and the mating surface of the cover at the location of theform-in-place gasket. ‘Thin’ refers to the thickness of an item, such asa gasket, and a ‘thin cross section’ references a cross section that isof limited thickness. The width of the gasket is defined as the distancefrom one side of the gasket material through the gasket material to theopposing side of the gasket material, proceeding on a line parallel tothe gasket surface but perpendicular to gasket's long axis at theparticular point where the length is being measured. ‘Narrow’ refers tothe width of an item, and a ‘narrow cross section’ references a crosssection that is of limited width. The length is the distance tracedaround the perimeter of the area, space, or chamber enclosed by thegasket. The length is typically much larger than the thickness or width.The gasket's long axis at any point is defined by the direction in whichlength is measured at that point. The cross section of the gasket refersto the area (or shape) of that portion of a plane through which thegasket passes, the plane being perpendicular to the long axis of thegasket (the axis along which length is determined). FIG. 3 schematicallyillustrates a cross section of a form-in-place gasket 34 formed on asubstrate 30 with a cover ready to be put into place on theform-in-place gasket 34 (move in the direction of the arrows 52). FIG. 3also schematically illustrates the meaning of thickness (denoted byarrows 54) and width (denoted by arrows 56). The length of aform-in-place gasket is dictated by the structure of the substrate, thecover, and of the area, space, or chamber defined by the gasket,substrate, and cover. The thickness of a gasket is generally dictated bythe choice of gasket material and method and/or conditions ofapplication of the gasket material (including amount of pressureapplied, if any, to squeeze the substrate and cover together). The widthof the gasket material may also be dictated by the choice of gasketmaterial and the method and/or conditions of application of the gasketmaterial (including whether the cover is applied before curing of thegasket material). The thickness and width of the gasket may be widelyvaried and may be selected based on the desired characteristics of thedevice being made.

Using the methods described herein, the thickness of the gaskets aretypically at least about 10 micrometers, more typically at least about15 micrometers, preferably at least about 20 micrometers, and thethickness may range up to about 25 micrometers in some embodiments, upto about 50 micrometers in other embodiments, and up to about 100micrometers, or even about 250 micrometers in still other embodiments.In larger scale devices (such as where assay chambers larger than about2 milliliters are contemplated) the thickness may be up to about 250micrometers in certain embodiments, up to about 500 micrometers in someembodiments, up to about 1000 micrometers or even up to about 2500micrometers in yet other embodiments. Using the methods describedherein, the width of the gasket is at least about 100 micrometers,typically at least about 150 micrometers, more typically at least about200 micrometers, at least about 250 micrometers in some embodiments,more preferably at least about 300 micrometers in certain embodiments,and the width may range up to about 250 micrometers in otherembodiments, or up to about 400 micrometers, or even up to about 500micrometers in other embodiments, or up to about 700 micrometers, oreven up to about 1000 micrometers in particular embodiments. In largerscale devices (such as where assay chambers larger than about 2milliliters are contemplated) the width may range up to about 1.5millimeters, typically up to about 3 millimeters, more typically up toabout 6 millimeters.

The thickness and/or width may be influenced by the characteristics ofthe gasket material (e.g. viscosity) and the conditions under which itis applied and/or cured, including whether a cover is in place (pressedwith the mating surface against the gasket material) during curing andhow much pressure is applied to the cover. The choice of gasket materialwill thus influence the physical dimensions of the gasket, and thedesired physical dimensions of the gasket will influence the choice ofgasket material. A range of gasket thickness may be obtained by varyingthe process suitably, for example by varying the choice of gasketmaterial or the method used to apply the gasket material to thesubstrate.

Other embodiments may use a gasket material, e.g. either a self-levelingor a non-slumping gasket material, to form a fluid containmentstructure, e.g. a well, on a substrate by depositing the gasket materialonto a substrate in a configuration in which the gasket material is atthe perimeter of an interior area of the substrate (defining theinterior area), the gasket material and interior area providing a wellthat may be used as to confine a fluid to the interior area. In aparticular embodiment, an analysis component is in operable relation tothe fluid containment well, such that in use, while the fluidcontainment well operates to confine the sample fluid, the analysiscomponent is used in the analysis of the sample fluid. In oneembodiment, the present invention provides a method for making aform-in-place gasket on a gasket surface on a substrate so that theform-in-place gasket is adjacent to a biochemical assay site. In thisembodiment, the method comprises making the form-in-place gasket havinga desired configuration by depositing a suitable gasket material in apredetermined configuration onto the gasket surface at a site adjacent abiochemical assay site, and then curing the gasket material to providethe finished form-in-place gasket having the desired configuration. Thegasket material is selected to provide a finished gasket that isflexible, inert to the conditions under which the biochemical assay isconducted, and having a very thin cross section. In this regard,“biochemical assay site” references an analysis site at which abiochemical assay is intended to occur. A biochemical assay is an assaythat is intended to analyze an analyte containing a biomolecule or whichuses a biomolecule to analyze an analyte, such as a hybridization assayusing a bioarray to analyze a sample fluid. The biochemical assay sitegenerally includes at least one analysis component, e.g. a biochemicalreagent, or is adapted to receive at least one analysis component. Thebiochemical assay site may optionally include a sensor for enablingsensing of results of the biochemical assay. In one embodiment, themethod comprises making the form-in-place gasket having a desiredconfiguration by depositing a suitable gasket material in apredetermined configuration onto the gasket surface at a site which,upon further assembly, will be adjacent a biochemical assay site, andthen curing the gasket material to provide the finished form-in-placegasket having the desired configuration.

The invention provides for an assay chamber associated with or includinga biochemical assay site. The assay chamber includes a substrate thathas a gasket surface with a form-in-place gasket on the gasket surface.The assay chamber also includes a cover having a mating surface that iscomplementary to the gasket surface and that can be placed adjacent thesubstrate to form a fluid-tight assay chamber. To form an assay chamber,the gasket surface should be adapted to fit the mating surface of thecover. In certain embodiments the assay chamber further includes atleast one analysis component (e.g. an array of immobilizedoligonucleotides) necessary for performing a biochemical assay, such as,e.g. a binding reaction between an immobilized oligonucleotide and acomplementary oligonucleotide in the sample solution. In one embodiment,the biochemical assay chamber is formed by a process where the gasketmaterial is first cured to form the form-in-place gasket, and then acover is placed adjacent the substrate with the form-in-place gasketdisposed between the cover and the substrate. In another embodiment, thecover is first placed on the gasket material, and then the gasketmaterial is cured. The assay chamber may have an alternate configurationthat provides for the biochemical assay; for example, the gasket surfacemay be on the cover, with the form-in-place gasket formed on the coverrather than on the substrate—in such case the complementary surface ison the substrate. Assay chambers according to the current invention mayhold any volume of fluid that the assay chambers may be designed tohold. In some embodiments the volume of the assay chamber may be atleast about 0.1 microliter, or at least about 1 microliter, or at leastabout 10 microliters, or at least about 100 microliters, depending onthe desired design of the assay chamber. In some embodiments the volumeof the assay chamber may up to about 100 microliters, or up to about 1milliliter, or up to about 10 milliliters. In designs for handlinglarger amounts of fluid, the volume of the assay chamber may be up toabout at 101 iters or more, depending on the desired design of the assaychamber.

It is commonly known that some analytes cause absorption of light ofcertain wavelengths, and some analytes produce changes in fluorescenceof a detector molecule. Thus, it is contemplated that both lightabsorption and fluorescence can be used for sensing the presence andconcentration of certain analytes. As used herein, the term “lightinteraction” refers to light absorption, fluorescence, phosphorescence,luminescence, and the like, occurring at an analysis site. In someembodiments, analysis of an analyte in the assay chamber uses a lightdetector associated with the assay chamber to measure the lightinteraction. It is to be understood that other types of lightinteraction may be monitored (as with a detector) to determine thepresence and concentration of analytes in view of the presentdisclosure.

In embodiments including arrays in the assay chambers, well know artprovides teaching for manufacture and use of the arrays, and it iswithin ordinary skill to use and to adapt this art to provide arrays onsubstrates such as are used herein in connection with the invention.Such art includes U.S. Pat. No. 6,242,266 to Fisher, U.S. Pat. No.6,232,072 to Schleifer et al., U.S. Pat. No. 6,180,351 to Cattell, U.S.Pat. No. 6,171,797 to Perbost, U.S. Pat. No. 6,323,043 to Caren et al.,U.S. Pat. No. 5,599,695 to Pease et al., U.S. Pat. No. 5,753,788 toFodor et al., U.S. Pat. No. 6,329,143 to Stryer et al., U.S. Pat. No.6,371,370 to Sadler et al., 5,721,435 to Troll, U.S. Pat. No. 5,763,870to Sadler et al., and U.S. Pat. No. 6,403,957 to Fodor et al. In certainembodiments, the analysis site may be adapted for use with commerciallyavailable optical scanning systems, examples of which are described inU.S. Pat. Nos. 5,837,475, 5,760,951 (confocal scanner) and U.S. Pat. No.5,585,639 (off axis scanner), all incorporated herein by reference.Typical scanning fluorometers are commercially available from differentsources, such as Molecular Dynamics of Sunnyvale, Calif., GeneralScanning of Watertown, Mass., Hewlett Packard of Palo Alto, Calif. andHitachi USA of So. San Francisco, Calif. Analysis of the data, (i.e.,collection, reconstruction of image, comparison and interpretation ofdata) is performed with associated computer systems and commerciallyavailable software, such as IMAGEQUANT™ by Molecular Dynamics orGENECHIP™ by Affymetrix of Santa Clara, Calif. Typically, a laser beamor other light source is used to illuminate the analysis site, whichexcites fluorescent labels used in the assay. The fluorescence signal isdetected by a detector and processed by a computer to determineinformation about the analyte, such as concentration, identity, and/orbinding affinity.

In varying embodiments, different arrangements wherein the array isinterrogated without removing the array from the chamber may easily beenvisioned, for example the cover is made of transparent glass orplastic and the array reader is adapted to interrogating the arraythrough the transparent glass or plastic cover. In such an embodimentthe chamber may include an inlet port and an outlet port, to allowintroduction and removal of, e.g., target solution (the analyte),rinsing solution, or other reagents.

FIG. 4 depicts an embodiment with multiple fluid containment structuresand multiple biochemical assay sites. In certain embodiments thebiochemical assay sites comprise an array 60, for example a bioarray,that is to be used in a biochemical assay. It should be understood thatthe biochemical assay (such as, in an embodiment, a bioarray) may bemanufactured directly onto the substrate surface 36 or may bemanufactured on an alternate material that is then immobilized on thesubstrate, for example in a well or a depression in the substratesurface. The invention will be herein described as it relates to arrays60 on the substrate 30, but it should be apparent that any suitablebiochemical assay may be substituted in place of the array 60 by one ofskill in the art given the disclosure herein. A skilled practitionerwill be able to adapt methods of manufacturing bioarrays that are knownin the art to provide one or more bioarrays on the substrate. Such knownmethods are described in U.S. patents and other references cited herein.

Referring now to FIG. 4, the invention as described herein may bepracticed in an embodiment wherein one or more arrays 60 (e.g.bioarrays) are disposed on the substrate surface 36 of a singlesubstrate 30. The substrate surface 36 further has a plurality ofform-in-place gaskets 34 disposed thereon, each form-in-place gasket 34encircling one or more arrays 60. One or more covers (not shown) may bedisposed closely adjacent the substrate and contacting the form-in-placegaskets 34 to form a fluid tight seal around each array 60. In someembodiments, ports (e.g. inlet and/or outlet) may be present, forexample in the cover, allowing fluidic assess to the assay chamberdefined by the substrate surface, the cover, and the form-in-placegasket. In the embodiment depicted in FIG. 4, the arrays 60 produced ona given substrate 30 need not be identical and some or all could bedifferent from the other arrays 60 present on the given substrate 30.

In one embodiment, about 2 to 100 of such bioarrays can be fabricated ona single substrate (such as glass). In such embodiment, after thesubstrate has the biomolecules on its surface, the substrate may be cutinto substrate segments, each of which may carry one or two or morebioarrays. In such cases gasket material may be deposited inpredetermined configurations onto the substrate before and/or after thesubstrate is cut into substrate segments. The narrow gaskets that formaround the individual areas of a multiple array substrate would beeasier to form a seal that a traditional single gasket with multipleopenings. Where a pattern of bioarrays is desired, any of a variety ofgeometries may be constructed, including for example, organized rows andcolumns of bioarrays (for example, a grid of bioarrays, across thesubstrate surface), a series of curvilinear rows across the substratesurface (for example, a series of concentric circles or semi-circles ofbioarrays), and the like. One or more analysis components may beassociated with each bioarray. The gasket material may, in oneembodiment, make a closed loop around each bioarray as shown in FIG. 4.In other embodiments, the predetermined configuration for applying thegasket material may leave one or more gaps (ports, or inlets andoutlets) as in FIG. 5.

In some embodiments the invention provides multiple arrays on a singlesubstrate, wherein multiple assay chambers are formed as describedherein by one or more covers disposed over the single substrate, whereina form-in-place gasket is disposed between the cover(s) and substrate.An exemplary embodiment is illustrated in FIG. 5 showing a multiplearray substrate with form-in-place gaskets 34. A substrate 30 with asubstrate surface 36 has a plurality of individual arrays 60 disposedthereon. The substrate 30 also has form-in-place gaskets 34 disposedalongside the individual arrays 60 extending from an inlet site 62 to anoutlet site 64, as shown in FIG. 5. In some embodiments theform-in-place gasket may be formed onto one or more covers adapted tobeing disposed on the substrate. In certain embodiments both thesubstrate and the cover will have form-in-place gaskets. When a cover(not shown) is placed in position on the substrate 30, a series ofparallel assay chambers is formed, each assay chamber defined by thesubstrate, the cover, and the form-in-place gaskets. Each assay chamberwill include one or more arrays, depending on the design. Each assaychamber has an inlet defined by the cover, form-in-place gasket 34, andsubstrate 30 at inlet site 62. Similarly, each assay chamber has anoutlet defined by the cover, form-in-place gasket 34, and substrate 30at outlet site 64. Fluid may be introduced into the assay chamber viathe inlet, and the outlet serves to vent the assay chamber and/orprovide a way for the fluid to leave the assay chamber. It will beappreciated that further liquid handling structures may be included,using the deposited gasket material or other well known manufacturingtechniques to include e.g. fluid reservoirs, flow conduits, vents,mixing structures, and the like. It will also be appreciated thatsimilar arrangements of elements are within the intended scope of theinvention, e.g. a substrate supporting multiple arrays disposed thereonmay be mated against a cover having a form-in-place gasket to formmultiple assay chambers.

The embodiment shown has arrays 60, inlets, and outlets equidistantlydisposed across the substrate, that is, they are spaced at uniformintervals. Assay chambers are provided by a cover disposed adjacent thesubstrate with the form-in-place gasket between the substrate and thecover, and the assay chambers are spaced at uniform intervals. Asindicated in FIG. 5 (at the arrows 68), the arrays are disposed on 4.5mm centers, which is compatible with the form factor of microassayplates that have a 16×24 array of wells (e.g. 384 well microtiterplates) and also compatible with fluid handling equipment (e.g. anautomated fluid dispensing system) designed to be used with suchmicroassay plates. The multiple array substrate may be fabricated inother configurations, for example, with the arrays disposed on 9 mm or2.25 mm centers on the substrate, in which case the multiple arraysubstrate would be compatible with the form factor of microassay platesthat have a 8×12 array of wells (e.g. 96 well microtiter plates) or a32×48 array of wells (e.g. 1536 well microtiter plates), respectively,and also compatible with fluid handling equipment (e.g. automated fluidhandling equipment) designed to be used with such microassay plates. Insome embodiments, the multiple array substrates may include more arrayson a substrate, e.g. from about 8 or 12 arrays per substrate or evenfrom about 16 or 24 arrays per substrate or even from about 32 or 48arrays per substrate, up to about 96 arrays per substrate, or even up toabout 384 arrays per substrate, or even up to about 1536 arrays persubstrate, or even more. In some embodiments, the substrates may bestacked such that the backside of a first substrate may serve as thecover for a second substrate, with a form in place gasket formed oneither the backside of the first substrate or the surface of the secondsubstrate. Stacking multiple substrates in such a fashion would providea multiple array unit having many individual assay chambers, each withone or more arrays. A similar unit would be formed by alternatelystacking multiple array substrates with covers. The multiple arraysubstrates described thus may be used to perform multiple arrayhybridization assays in a large scale parallel format, greatlyincreasing throughput as compared to individual (or small multiple, i.e.less than about 3 arrays per substrate) array substrates.

Still other embodiments are illustrated in FIGS. 6 and 7. In FIG. 6,form-in-place gaskets 34 are disposed on a substrate 30 in aconfiguration providing for a plurality of assay chambers arranged inranks 72, 74, wherein the assay chambers in a given rank areequidistantly disposed (spaced at uniform intervals). Note that thearrays 60 on the substrate 30 are equidistantly disposed at a differentuniform interval (arrows 76) than the uniform interval (arrows 78) ofthe inlets and outlets, due to the arrangement into ranks 72, 74. InFIG. 7, the form-in-place gaskets 34 are disposed on a substrate 30 in aconfiguration providing for a plurality of assay chambers arranged inranks 72, 74, wherein the assay chambers are arranged “in series”between the ranks such that an assay chamber in the first rank is influid communication with an assay chamber in the second rank. In anembodiment having assay chambers in series, the sample fluid may bepumped into the first assay chamber, then more fluid may be introduced,pushing the sample fluid into the second assay chamber, then the fluidmay be withdrawn from the first chamber (reverse flow) such that thesample fluid is transferred from the second chamber back into the firstchamber. This may be repeated several times to provide mixing of thesample fluid and improved contact between the sample and any analysiscomponent (e.g. an array) associated with the first assay chamber.

To form the array, the biomolecule is typically applied to a surface,e.g. the surface of the substrate, by spotting, using pipettes, pins,inkjets, or the like. Methods of depositing materials onto a planarsurface are known, including loading and then touching or tapping a pinor capillary to the surface (U.S. Pat. No. 5,807,522 to Brown et al.;U.S. Pat. No. 6,110,426 to Shalon, et al.); employing an array of pinsor capillaries to transfer an array of droplets to a surface (Lehrach,et al., “Hybrididization Fingerprinting in Genome Mapping andSequencing,” in Genome Analysis, Vol. 1, pp. 39-81 (1990, Davies andTilgham, Eds., Cold Spring Harbor Press)). Ink jet technology may beused to spot biomolecules and other reagents on a surface, for example,using a pulse jet such as an inkjet type head to deposit a droplet ofreagent solution for each feature. See, for example, PCT publications WO89/10977, WO 95/25116 and WO 98/41531, and elsewhere. Still othermethods and apparatus for fabrication of polynucleotide arrays aredescribed in, e.g. U.S. Pat. No. 6,242,266 to Schleiffer et al., whichdescribes a fluid dispensing head for dispensing droplets onto asurface, and methods of positioning the head in relation to the surface.Other methods include those disclosed by U.S. Pat. No. 6,180,351 toCattell; U.S. Pat. No. 6,171,797 to Perbost; Gamble, et al., WO97/44134;Gamble, et al., WO98/10858; Baldeschwieler, et al., WO95/25116; and thelike. Other methods can also be used to deposit biomolecules on thesurface including those employing photolithographic techniques forforming arrays of moieties, such as described in U.S. Pat. Nos.5,807,522; 5,143,854; 5,405,783; and 5,744,305. A number of other knownmethods are available and may be used for depositing the biomolecules ona surface. Modifications of these known methods within the capabilitiesof a skilled practitioner in the art as well as other methods known tothose of skill in the art may be employed.

In one embodiment, the bioarray has array features comprisingoligopeptides deposited on the surface of the substrate. In otherembodiments, other biomolecules, such as polypeptides, oligonucleotides,polynucleotides, or known analogues or derivatives of any of theforegoing, or combinations of any of the foregoing, are deposited on thesubstrate. Any given array feature can have the same or a differentbiomolecule or combination of biomolecules compared to any other givenarray feature. Biomolecules may be derived from natural sources (e.g.isolated from cellular material) or may be synthetic. Examples ofbiomolecules include antigenic epitopes, fragments of antibodies orother proteins, polysacharrides, cDNAs, and RNAs.

The biomolecules may bind directly to the substrate surface or may bindvia an intermediate moiety upon the surface, e.g. a bifunctional linkermolecule or other surface treatment. Polynucleotides may be bound to thesurface by irradiating with UV light, during which the polynucleotidescovalently attach to the surface, typically via an intermediate moiety,presumably, by non-specific, free-radical cross-linking. Chemicalmethods for covalently binding biomolecules in an array format tosubstrate surfaces are known in the art and may be employed by one ofordinary skill in the art.

In bioarray fabrication, the quantities of biomolecule available areusually very small and expensive. Additionally, sample quantitiesavailable for testing are usually also very small and it is thereforedesirable to simultaneously test the same sample against a large numberof different probes on a bioarray. Therefore, one embodiment of theinvention provides for fabrication of bioarrays with large numbers ofvery small, closely spaced array features. Arrays may be fabricated witharray features that may have diameters (assuming a round spot) in therange from a minimum of about 10 micrometers to a maximum of about 1.0cm. In embodiments where very small spot sizes or array feature sizesare desired, material can be deposited in small spots whose width is inthe range about 1.0 micrometer to 1.0 mm, usually about 5.0 micrometersto 0.5 mm, and more usually about 10 micrometers to 200 micrometers.Interfeature areas will typically (but not essentially) be present whichdo not carry any biomolecule. It will be appreciated though, that theinterfeature areas could be of various sizes and shapes.

A bioarray may contain any number of array features, generally includingat least tens of array features, usually at least hundreds, more usuallythousands, and as many as a hundred thousand or more array features. Allof the array features may be different, or some or all could be thesame. Each array feature carries a predetermined biomolecule or apredetermined mixture of biomolecules, such as a particularpolynucleotide sequence or a predetermined mixture of polynucleotides.The array features may be arranged in any desired pattern, e.g.organized rows and columns of array features (for example, a grid offeatures across the substrate surface), a series of curvilinear rowsacross the substrate surface (for example, a series of concentriccircles or semi-circles of features), and the like. In embodiments wherevery small array feature sizes are desired, the density of features onthe substrate may range from at least about ten array features persquare centimeter, or preferably at least about 35 array features persquare centimeter, or more preferably at least about 100 array featuresper square centimeter, and up to about 1000 array features per squarecentimeter, or preferably up to about 10,000 array features per squarecentimeter, or perhaps up to 100,000 array features per squarecentimeter.

The substrate and the cover may take any of a variety of conformationsranging from simple to complex. Thus, the substrate could have generallyplanar form, as for example a slide or a plate, such as a rectangular-or square- or disc-shape. In many embodiments, the substrate will beshaped generally as a rectangular solid, having a length in the rangeabout 4 mm to 400 mm, usually about 4 mm to 150 mm, more usually about 4mm to 125 mm; a width in the range about 4 mm to 400 mm, usually about 4mm to 120 mm and more usually about 4 mm to 80 mm; and a thickness inthe range about 0.01 mm to 5.0 mm, usually from about 0.1 mm to 2 mm andmore usually from about 0.2 to 1 mm. In other embodiments the substratemay have larger dimensions. The substrate surface may be smooth orsubstantially planar, or have irregularities, such as depressions orelevations. The shape of the substrate may be selected according tomanufacturing, handling, and use considerations. The cover will beshaped to provide a mating surface that is complementary to the gasketsurface of the substrate such that the cover can be positioned againstthe form-in-place gasket to form a fluid tight seal. The cover may besmooth or substantially planar, or have irregularities, such asdepressions or elevations.

The process of the current invention may be employed on any substratehaving a surface to which the gasket material may bind. Preferredsubstrate materials provide physical support for the gasket material andendure the conditions of the deposition process and of any subsequenttreatment or handling or processing that may be encountered in the useof the substrate. Suitable substrates may have a variety of forms andcompositions and may derive from naturally occurring materials,naturally occurring materials that have been synthetically modified, orsynthetic materials. Examples of suitable substrate materials include,but are not limited to, nitrocellulose, glasses, silicas, teflons,metals (for example, gold, platinum, and the like), and ceramics(including aluminum oxide and the like), composites, and laminatesthereof. Suitable substrate materials also include polymeric materials,including polysaccharides such as agarose (e.g., that availablecommercially as Sepharose®, from Pharmacia) and dextran (e.g., thoseavailable commercially under the tradenames Sephadex® and Sephacyl®,also from Pharmacia), polyacrylamides, polystyrenes, polyvinyl alcohols,copolymers of hydroxyethyl methacrylate and methyl methacrylate,polyesters, including poly(ethylene terephthalate) and poly(butyleneterephthalate); polyamides, (such as nylons); polyethers, includingpolyformaldehyde and poly(phenylene sulfide); polyimides, such as thatmanufactured under the trademarks KAPTON (DuPont, Wilmington, Del.) andUPILEX (Ube Industries, Ltd., Japan); polyolefin compounds, includingABS polymers, Kel-F copolymers, poly(methyl methacrylate),poly(styrene-butadiene) copolymers, poly(tetrafluoroethylene),poly(ethylenevinyl acetate) copolymers, poly(N-vinylcarbazole),polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, andblends thereof, and the like. Certain polymeric materials that may beused for substrate materials include organic polymers that are eitherhomopolymers or copolymers, naturally occurring or synthetic,crosslinked or uncrosslinked. The cover may be formed from the sametypes of materials as given herein for the substrate.

The devices of the invention may also be fabricated from a “composite,”i.e., a composition comprised of unlike materials. The composite may bea block composite, e.g., an A-B-A block composite, an A-B-C blockcomposite, or the like. Alternatively, the composite may be aheterogeneous combination of materials, i.e., in which the materials aredistinct from separate phases, or a homogeneous combination of unlikematerials. As used herein, the term “composite” is used to include a“laminate” composite. A “laminate” refers to a composite material formedfrom several different bonded layers of identical or differentmaterials. Other preferred composite substrates include polymerlaminates, polymer-metal laminates, e.g., polymer coated with copper, aceramic-in-metal or a polymer-in-metal composite.

The substrate surface may optionally exhibit surface modifications overa portion or over all of the surface with one or more different layersof compounds that serve to modify the properties of the surface in adesirable manner. Such modifications include: inorganic and organiclayers such as metals, metal oxides, conformal silica or glass coatings,polymers, small organic molecules, hetero-bifunctional linkingmolecules, and the like. Polymeric layers of interest include layers of:polypeptides, proteins, polynucleotides or mimetics thereof, e.g.peptide nucleic acids and the like; polysaccharides, phospholipids,polyurethanes, polyesters, polycarbonates, polyureas, polyamides,polyethyleneamines, polyarylene sulfides, polysiloxanes, polyimides,polyacetates, and the like, where the polymers may be hetero- orhomopolymeric, and may or may not have separate functional moietiesattached thereto, e.g. conjugated.

Components of the assay chambers (e.g. covers, substrates, etc.)according to the present invention can be fabricated using anyconvenient method, including, but not limited to, molding and castingtechniques, embossing methods, surface machining techniques, bulkmachining techniques, and stamping methods. Further, injection moldingtechniques well known in the art may be useful in shaping the materialsused to produce components according to the instant invention.

Typical use of the system is given in the examples which follow, whichillustrate various embodiments according to the present invention butshould not be construed to limit the invention as claimed.

EXAMPLES

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of device manufacture, materialmolding and shaping, applying coatings, synthetic organic chemistry,biochemistry, molecular biology, and the like, which are within theskill of the art. Such techniques are explained fully in the literature.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how toperform the methods and use the compositions disclosed and claimedherein. Efforts have been made to ensure accuracy with respect tonumbers (e.g., amounts, temperature, etc.) but some errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, temperature is in ° C. and pressure is at or nearatmospheric. Standard temperature and pressure are defined as 20° C. and1 atmosphere.

Forming the Form-in-Place Gasket

An example of a process of making a form-in-place gasket using acontrolled dispensing system, in this example an adhesive dispensingmachine, is now described. The dispensing system has a computercontrolled positioning system to control the position and feed rate of adispensing tip. The computer may be programmed to control delivery ofgasket material from the dispensing tip. A suitable system is theAutomove 403 and is available from Asymtek (Carlsbad, Calif.). The typeof gasket, whether thin or thick, and the shape or profile, willdetermine the types of gasket material appropriate for the application.For thin gaskets, one would select a low viscosity, self-levelingmaterial. One would select a small diameter orifice dispense tip, in the25 to 29 gauge range; a small diameter dispense tip keeps the amount ofmaterial dispensed and the diameter of the bead small. When using smalldiameter tips, the dispense rate is also low requiring a slow velocityof the dispense tip. Experimenting has shown that dispense tipvelocities of more than about 0.01 inches per second and less than about10 inches per second are suitable for the thin gaskets, though thedispense tip velocity may extend higher or lower in certain embodiments.For the thin gaskets, it is desirable to tightly control the paralleltravel of the dispense tip with respect to the substrate surface wherethe gasket will be placed as well as the height of the dispense tipabove the surface. Since the typical distance of the tip above thesurface is between 0.002 and 0.005 inches, changes in the height due tounparallel travel of the tip is significant. For thicker gaskets,adjustments would be made, including one or more of, e.g. a largerdiameter dispense tip, a non-slumping gasket material, altering theangle and height of the dispense tip relative to the gasket surface,etc. The amount of gasket material dispensed and the rate at which thegasket material is dispensed affect the uniformity of the gasket height(thickness). The simplest dispense system is the pressure regulatedsyringe. The feed rate is determined by the diameter of the dispensetip, its length, viscosity of gasket material and the pressure appliedto the reservoir. The gasket material is transferred from its containerto a syringe barrel. The dispense tip end is capped closed and thesyringe is centrifuged to eliminate air pockets in the column of gasketmaterial in the syringe. Air gaps in the gasket material inside thesyringe cause problems during dispensing. First, if an air gap reachesthe dispense tip, this disrupts the flow of gasket material and causesdefects in the gasket as it is forming. Second, air gaps arecompressible and they will cause a change in flow rate (also called feedrate) of the gasket material. After the centrifugation, a plunger isplaced in the syringe barrel and pushed until it contacts the gasketmaterial. The cap at the dispense end of the syringe is removed and adispense tip is attached. The syringe is placed on the dispense systemand attached to the pressure source. The pressure regulator is set tothe proper dispense pressure for the application. Pressure is applied tothe syringe to prime the dispense tip. The pressure is kept on until asteady stream of gasket material is being dispensed. One could collectthe gasket for a set time and weigh it to check the feed rate. Thepressure is adjusted for proper feed rate. In most cases, the dispensesyringe and tip are disposable and as a result are usually at adifferent height and position with respect to the dispense system. Usingthe calibration tools, the tip position is measured and adjusted.

Each type of gasket, shape, height and part has a different program onthe computer controlled dispensing system. The proper program is loadedfrom the computer to the operating software. The substrate is held inplace so as to have a reference position on the dispense system. Inaddition, the dispense system can reference to the substrate using anoptical measurement system such as a camera to adjust for parts thatvary slightly. A convenient hold down mechanism is vacuum fixture. Whilethe vacuum is off, the substrate is placed in position on the fixture.The vacuum is turned on and then held in place. After the substrate isin place and secure, a test run is performed. After the test run, thesubstrate is evaluated for correctness. This is usually a visualinspection to ensure a complete gasket and that the shape and profileare correct within limits determined by the application. One or moretest runs can be performed for this evaluation. If corrections areneeded, one or more of the parameters are adjusted. Usually, if thecalibration is done correctly for the dispense tip position and the feedrate of the gasket material is within proper range, no furtheradjustments are required. The process is now ready to start producinglarge numbers of gaskets. To ensure consistent gaskets, monitoring forgasket shape and height is required. The feed rate can also be monitoredafter a set number of parts have been fabricated to check for changes.Again, adjustments can be made for any changes.

Preparing Sample for Array Hybridization:

The form-in-place gasket can be used with any kind of reaction that canbe adapted to use chambers such as those described herein.Oligonucleotide arrays and protein arrays are specific applications inwhich a chamber includes or encloses a “probe”, otherwise known as acapture agent, attached to a surface, such as glass. The form-in-placegasket can be on the array glass or on the cover that creates the otherhalf of the chamber. In this example, illustrated in FIG. 8, a cover 40has a gasket surface 32 with a form-in-place gasket 34 formed on thegasket surface. The cover 40 with the form-in-place gasket 34 forms ashallow well that can hold an aliquot of fluid, e.g. of the targetsample. The cover 40 is adapted to having the array substrate 30 placedover the cover 40 (following arrows 70), thus forming an assay chamberin which the array 60 may be contacted the fluid in the assay chamber.The thickness of the form-in-place gasket can be varied depending onsystem and experimental design. The layout, size and thickness of theform-in-place gasket depends on the volume of sample desired and theparticular application.

Specifically, an 8500 feature array with single stranded oligonucleotideprobes was used. Conditions for hybridization assays are well known inthe literature and can be adapted by one of ordinary skill in the art tomeet the design considerations of the particular assay used. In thisexample, the following components were placed in a 1.5 mL nuclease-freemicrocentrifuge tube: 1.25 μg cyanine 3-labeled linearly amplified cRNA,1.25 μg cyanine 5-labeled linearly amplified cRNA, and 25 μL 10× ControlTargets. Control targets are complementary targets that are spiked(added) at a known concentration; the probes for the control targets areusually on the border of the array—these spots help the analysissoftware find the borders. Then, nuclease-free water was added to make avolume of 125 μL. Then, 2× hybridization buffer (150 mM LiMES (pH 6.1),612 mM LiCl; 1.0% octylphenol ethylene oxide condensate (tradenameTriton X-100®), 1.5% lithium lauryl sulfate, 6 mM EDTA) was added tobring the volume to 250 μL. This is what was used as the target sample.The target sample was vortexed briefly to mix it, and then the samplewas spun down in a microcentrifuge. It will be appreciated that othervolumes of target sample can be used as well, and that such variationsin system and experimental design lie within ordinary skill.

Loading the Sample Onto a Form in Place Gasket and Hybridizing:

In order to load the biological sample, the cover with the form-in-placegasket was placed on the work bench. The target sample was placed,either by pipetting or by some other means, into the well formed by thegasket on the cover. The array substrate was placed over the cover withthe active (array) side down, so that the array would interact with thetarget sample (as indicated by the arrows 70 in FIG. 8). The assemblywas clamped together so that it was secure in the assembly clamp. Theassay chamber assembly (array substrate plus gasket/cover plus targetsample) was placed on the rotator rack in the hybridization oven (ovenwith rotating rack mechanism is from Robbins Scientific, Sunnyvale,Calif.), at 60° C. Each assay chamber assembly was clamped on its sideand was rotated end-over-end on the rotator rack to achievemixing/stirring of the target sample in the assay chamber. Thehybridization rotator rack was set to rotate at about 4 rpm. Other assaychamber assemblies were similarly prepared and placed in the rotatorrack. The hybridization was conducted at 60° C. for 17 hours. Inaccordance with the invention, the conventional hybridization solutionsand processes for hybridization can be used, such as those described inSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, Ed. 2nd, 1989, vol. 1-3,incorporated herein by reference. Conditions for hybridization typicallyinclude (1) high ionic strength solution, (2) at a controlledtemperature, and (3) in the presence of carrier DNA and detergents anddivalent cation chelators, all of which are well known in the art.Increased stringency is achieved by elevating the temperature,increasing the ratio of co-solvents, lowering the salt concentration,and the like.

The form-in-place gasket can also be used at most temperatures up to thehighest specified temperature of the gasket material. The gasket alsoholds a seal in a centrifugal field, and may be used with a rotatorwhich acts as a centrifuge to impart a centrifugal force to enhancemixing in the assay chamber. Different rotating speeds can be used. Incertain embodiments, no rotation may be needed or used. This exampledescribes loading the sample manually (via pipetting), but the assaychamber incorporating the form-in-place gasket is also amenable toautomation.

Disassembly, Washing, Drying and Scanning the Array:

Before the incubation was finished, three staining dishes were prepared.Each held about 250 mL of solution. Wash Solution 1 (6×SSC, 0.005%Triton X-102) at 60° C. and a slide rack were placed in the firststaining dish. Wash Solution 1 (at room temperature, enough to cover aslide rack) and a magnetic stir bar were added to the second dish. Thethird dish was placed into a container filled with ice (a Pyrex loaf panis well-suited to this purpose). A magnetic stir bar and enough WashSolution 2 (0.1×SSC, 0.005% Triton X-102) at 4° C. to cover a slide rackwere placed into the third staining dish. The ice in the outer containerwas replenished as needed to keep the solution as cold as possible. Asingle assay chamber assembly was removed from the oven and inspected todetermine if bubbles formed during hybridization, and if all bubbleswere rotating freely. The cover, with the gasket, and the arraysubstrate were removed from the assembly clamp but kept together, makingsure the fluid did not leak out.

The two slides were placed in the slide rack in the first staining dish,which is filled with 60° C. Wash Solution 1. The assay chambers weredisassembled by separating the slides while they were immersed insolution. A thin object slid between the slide and cover aided inseparating the two pieces of glass. Disassembly of the chamber whileimmersed provides an advantage of quickly diluting the sample fluid,resulting in lower background signal. The cover (with the gasket) wasremoved from the slide rack and put aside. These steps were repeated forall remaining assay chamber assemblies. The slide rack with the arrayswas quickly transferred to the second staining dish (Wash Solution 1 atroom temperature) and set over a magnetic stir plate to stir at mediumspeed. The slides were washed for 10 minutes at room temperature. Theslide rack was then transferred to the third staining dish, which is onice. The dish was placed on a magnetic stirring plate set to mediumspeed. The slides were washed for 5 minutes at 0 to 4° C. Then the sliderack was removed from dish and placed directly into a centrifuge to drythe slides at 1000 RPM for 3 minutes. The slides were loaded into ascanner, and fluorescence intensities were measured. Any effectivemethod of array interrogation may be used, including various methodsknown to those of skill in the art. After scanning, slides were storedin polypropylene slide boxes without cork or foam inserts, in a vacuumdesiccator or a nitrogen purge box, in the dark.

The above example describes the process of using the form-in-placegasket in forming an assay chamber for array hybridization. It replacestwo prior different prior hybridization technologies—the “large volumehybridization method” and the “coverslip” method. The large volumehybridization method required assembling a chamber that consisted of amolded plastic backing part with holes that septa fit into. The arrayswere placed into a stainless steel holder then the plastic backing wasplaced on top. A rubber, square, O-ring was fit on top of the plasticbacking to give it some compliance. Then the top of the stainless steelchamber was placed on the O-ring. The two stainless steel assembly partswere secured together with 6 screws. These screws had to be tighteneddown with a screwdriver. Then the two septa per array were inserted intothe holes in the plastic molded backing part. After assembly of theassay chamber, the sample still had to be inserted into the assaychamber, where it would contact the array. This was done by placing asyringe needle into one septum to vent the chamber while the syringeneedle with the sample was placed into the other septum and the samplewas injected.

The other prior method of array hybridization used the “coverslip.” Inthis method the scientist placed the array on the work bench, pipettesthe sample onto the array and then places a coverslip on top. Thismethod is highly error prone, since the coverslip can move around easilybecause it just “floats” over the sample solution. The results fromcoverslip hybridizations were also very unrepeatable since coverslipsbent easily and bowed. This non-uniformity caused different parts of thearray to have different signals because of the varying height of thesample above each part of the array. Also, during the relatively warmtemperatures used for the hybridization experiments, the sample solutionwould evaporate from around the edge of the coverslip, adding to thenon-repeatability of the results.

The form-in-place gasket as described herein removes the need fornon-compliant molded parts, syringes, septa and the use of the screwdriver. It also eliminates problems associated with the coverslipmethod.

Multiple Array Format:

The invention also provides for the use of form-in-place gasketfabrication to construct a multiple array substrate wherein arrays on asingle substrate are separated by form-in-place gaskets resulting in one(or more) arrays per assay chamber (also called “assay channel”, in thisexample). A series of arrays are prepared using a standard 1×3 inchmicroscope slide as the array substrate. The arrays are disposed on thearray substrate on 4.5 mm centers. Multiple assay channels areconstructed by applying beads of silicone gasket material from one edgeof the slide to the other along the short dimension of the arraysubstrate. The layout of such a slide is as shown in FIG. 5. After thesilicone gasket material is applied to the array substrate, anotherglass slide (the cover) is placed on top, and the silicone is allowed tocure. Assay chambers (“assay channels”) are thus formed in the spacebetween the array substrate and the cover and between the beads ofsilicone. Depending on the choice of silicone gasket material, thethickness of the gasket (“height” of the assay chamber) may range fromabout 25 micrometers to about 200 micrometers. Given this range, thevolume for the above type of assay chamber is about 2 to about 15microliters. Other volumes are possible by varying the design, as shouldbe apparent.

Sample integrity is maintained by an air gap channel (feature 66 in FIG.5) between each assay chamber. If sample leaks around the inlet, theoutlet, or past a gasket forming the assay chamber, it is drawn into theadjacent air gap channel. If the sample is not completely drawn into thecapillary (assay channel) and excess remains at the opening, any excesswider than the opening is drawn into the adjacent air gap channel.

The multiple array substrate with the cover in place forms amulti-channel microarray. An additional feature can be added: at theopenings (the inlets and outlets, or ports) on the edge between the twoglass slides—another bead of silicone can be dispensed to form a gasket.This additional gasket is oriented on a plane that is not coincidentwith the substrate and can define further liquid handling structures,e.g. an interface port. The additional gasket disposed on the edge ofthe multi-channel microarray (the interface port) can then be used as amake and break seal (a reusable seal) for operations such as sampleintroduction, mixing, and washing. Such operations would be conducted atone or more “stations”, or fluid handling devices adapted to interfacewith the multi-channel microarrays. Such stations may be constructed bythose of skill in the art given the description herein of multi-channelmicroarrays. By having the gasket on the disposable multi-channelmicroarrays instead of the station, cross contamination betweenexperiments will be minimized.

Filling the Chamber With Sample:

In one example, the assay chamber, formed with the silicone beadsbetween the array substrate and the cover, is approximately 50 micronsin height. This assay chamber is essentially a capillary, and the liquidsample will be drawn into the capillary. Several techniques can beemployed to apply the sample to the opening (the inlet) on the end ofthe sandwiched slides. First, is a manual technique. Sample is aspiratedinto a disposable pipette, then the tip is place at the opening and thesample slowly dispensed as the capillary is filled.

Second, a flexible bottom microtiter plate can present a drop to theopening of the capillary (the inlet) and the sample will be drawn intothe channel. A multi-channel pipette can perform this operation inparallel. The automation of the operations is possible if the channelsare at a standard microtiter plate spacing. That would be 9 mm for a 96well plate, 4.5 mm for a 384 well plate, and 2.25 mm for a 1536 wellplate. Other possibilities include pumping the sample into the capillaryor sucking the sample into the capillary by a vacuum applied to theopposite side (the outlet).

Hybridization:

After the sample in a hybridization buffer has been drawn into thechannel, a standard hybridization can be performed. The multi-channelmicroarray can be placed in a humid chamber at the appropriatetemperature. Evaporation is controlled by the humid environment. This issimilar to the cover slip hybridization procedure used now.

Mixing:

The sample can be mixed on the array during hybridization by alternatingpressure and vacuum on the opening of the capillary pumping the liquidback and forth across the array. Flow channels and/or mixing chambersand or mixing structures may advantageously be incorporated into thedesign of the device.

Washing:

Washing may be accomplished by exchanging the sample/hybridizationsolution with wash buffer. This can be automated with the multi-channelmicroarray by mating the openings with a wash station and having thewash solution either pumped into or drawn into the channel. Thetemperature of the wash solution can also be controlled. In manualprocedures, many array substrates are placed in the same wash buffer.This can cause cross contamination. By having each channel perform thewash operation independently, cross contamination isminimized/eliminated.

Drying:

After washing, the wash buffer must be removed from the array and dried.This can also be automated by mating the openings with a drying stationand either pressurizing the chamber with N₂ or by vacuum on the outletside.

Automation of Entire Process:

Because the channels are at the spacing of a standard micro titer plate,automation of a complete microtiter plate's worth (or even many plates'worth) of samples is possible. For example, a rack of multi-channelmicroarrays can be set up for analysis of one or more microtiter platescontaining samples. For each microtiter plate (e.g. a 96 well plate),there could be, e.g. twelve multi-channel microarrays with eightchannels per substrate that would match the spacing of the samples inthe corresponding plate. The automated machinery would pick up asubstrate, apply samples from a row in the microtiter plate, andcontinue with the hybridization process.

By providing spacing of the channels that is compatible with microtiterplates, the multiple array format allows automation for samplepreparation prior to hybridization. All of the sample prep includingisolation, amplification, labeling can be done in microtiter plates andthen interfaced with the multiple array format for hybridization andwashing.

As described in this example, the processes and devices according thepresent invention may provide one or more of the advantages describedherein, including the following: existing array manufacturing processesmay be easily adapted for use with form-in-place gasket techniques;multiple array formats are easily provided (as compared to currentapproaches only using one or two arrays per substrate or largersubstrates cut into smaller pieces with one array per piece); easyconversion to automation of sample introduction, hybridization, andwashing; interfacing with standard microtiter plate automationequipment; scales to batch automation from a single substrate to largescale at microtiter plate increments (e.g 8, 12, 16, 24, 96, 384, 1536).

The invention thus also provides for an assay chamber. The assay chamberincludes a substrate that has a gasket surface with a form-in-placegasket on the gasket surface. The assay chamber also includes a coverhaving a mating surface that is complementary to the gasket surface andthat can be placed adjacent the substrate with the mating surfaceagainst the form-in-place gasket to form a fluid tight seal. Essentiallysimilar alternate configurations are possible, e.g. in which the gasketsurface is on the cover and the mating surface is on the substrate. Theassay chamber typically includes at least one analysis component (e.g.an array of immobilized oligonucleotides) necessary for performing, e.g.a biochemical assay, such as a binding reaction between an immobilizedoligonucleotide and a complementary oligonucleotide in the samplesolution.

The invention thus also provides for a fluid containment structure. Thefluid containment structure includes a substrate that has a gasketsurface with a form-in-place gasket on the gasket surface. Theform-in-place gasket is disposed around and marks the perimeter of aninterior area on the substrate. The interior area and the form-in-placegasket define a well that is adapted for retaining a fluid. The shape ofthe interior area may be altered depending on the desired use byaltering the configuration of the form-in-place gasket. The fluidcontainment structure may be associated with or form a portion of ananalysis site where a sample fluid retained in the fluid containmentstructure may be analyzed. The analysis site typically includes at leastone analysis component (e.g. an array of immobilized oligonucleotides)necessary for performing, e.g. a biochemical assay, such as a bindingreaction between an immobilized oligonucleotide and a complementaryoligonucleotide in the sample solution.

The invention provides a method of performing a hybridization assayusing an assay chamber that includes a form-in-place gasket. In oneembodiment the method includes mating a cover against a complementarysurface on a substrate, wherein the substrate has an array surface onwhich biomolecules are deposited in an array format. A form-in-placegasket is present on the cover, and when the cover is mated against thecomplementary surface an assay chamber is formed. The method may furtherinclude contacting the array surface with the solution to be tested,disassembling the chamber by removing the cover, processing the arraysurface, and interrogating the array surface using, e.g. an arrayreader. In alternate embodiments no disassembly is required, because theassay chamber is adapted to allow interrogation of the array surfacewithout disassembly, e.g the cover may have a transparent area allowinglight from the array reader to reach the array surface. In someembodiments, other configurations of assay chamber may be used, e.g. theform-in-place gasket may be present on the substrate surface and matedagainst a complementary surface on the cover, or the array surface mayform a portion of a separate array substrate that is held in placebetween the substrate and the cover.

The form-in-place gaskets of the current invention may be quite thinbecause the form-in-place gaskets do not have to be handled orpositioned in order to get the gasket properly positioned on theintended surface (because the gasket is formed on the surface). Thisminimizes problems of alignment, damage, and contamination that arose inprevious methods of applying pre-formed gaskets to surfaces. Earliermethods that use pre-formed gaskets require handling and positioning ofgaskets on the site where a fluid tight seal is desired. Problems withthe earlier methods arose when a very thin gasket was desired handlingand positioning very thin, pliable gaskets is difficult because thethinness makes the gasket delicate and easily damaged. The methodprovided herein is thus an advantageous solution to the noted problems.Where very small volumes of solution are desired in a biochemical assay,the thin, form-in-place gaskets described herein are advantageous forproviding biochemical assay chambers that are thin and as a consequencerequire only very small volumes of solution. While allowing a smallsample volume to be used for the biochemical assay, the form-in-placegasket keeps the substrate surface from coming into direct contact withthe surface of the cover. This can be beneficial where the analysiscomponent is located on the substrate surface and can be damaged byinadvertent contact with the cover.

The gasket material is typically deposited on a substrate and then iscured to form a fluid tight seal between the gasket material and thesubstrate surface. Prior methods of forming fluid tight seals usingpreformed gaskets typically require relatively thick gaskets and resultin a need to make a fluid tight seal between the cured gasket and thesubstrate and also between the cured gasket and the cover. In contrast,the present method results in a fluid tight seal between the gasketmaterial and the gasket surface as the gasket material is curing. Thisleaves only one fluid tight seal, between the cured form-in-place gasketand the mating surface on the cover, to be made upon placing the coveron the gasket. The cross section, or profile, of the form-in-placegasket may be dome-shaped, or rounded. A dome-shaped profile providesfor point contact with the mating surface of the cover, resulting inlower compressing forces needed to form a fluid tight seal.

While the foregoing embodiments of the invention have been set forth inconsiderable detail for the purpose of making a complete disclosure ofthe invention, it will be apparent to those of skill in the art thatnumerous changes may be made in such details without departing from thespirit and the principles of the invention. Accordingly, the inventionshould be limited only by the following claims.

All patents, patent applications, and publications mentioned herein arehereby incorporated by reference in their entireties.

1. An apparatus comprising a plurality of assay chambers, the apparatuscomprising, in order, a substrate, a form-in-place gasket, and a cover,wherein the form-in-place gasket, the substrate, and the cover togethersubstantially define a plurality of assay chambers, wherein theform-in-place gasket forms a permanent seal between the form-in-placegasket and the cover producing a plurality of fluid containmentstructures, each fluid containment structure housing at least one assaychamber; and the apparatus further comprises a plurality of arrays,wherein each array being in operable association with one or more of theassay chambers, wherein each fluid containment structure is contained onthe top by the cover, wherein each fluid containment structure iscontained on the side by the form-in-place gasket, wherein each fluidcontainment structure is contained on the bottom by the substrate,wherein the form-in-place gasket, the substrate, and the cover togetherfurther define a plurality of inlets and outlets, wherein the inlets andoutlets are through the form-in-place gasket in a plane parallel withthe substrate and the cover, wherein the inlet and the outlet aredisposed on opposite ends of each assay chamber, wherein each assaychamber is in fluid communication with an inlet and an outlet, whereinthe form-in-place gasket is between about 10 micrometers and about 250micrometers thick, wherein the form-in-place gaskets consists of gasketmaterials selected from at least the following: silicon sealants,urethanes, polysulfides, latex, and acrylic sealants, and wherein thecover and the substrate are made of different materials than theform-in-place gasket.
 2. The apparatus of claim 1, wherein the assaychambers are spaced at uniform intervals.
 3. The apparatus of claim 2,wherein the uniform interval is selected from the group consisting ofabout 4.5 mm, about 9 mm, and about 2.25 mm.
 4. The apparatus of claim3, wherein the inlets and outlets are in fluid communication with anexternal fluid dispensing system that handles multiple fluids inparallel.
 5. The apparatus of claim 4, wherein the form-in-place gasketis between about 10 micrometers and about 100 micrometers thick.
 6. Theapparatus of claim 4, wherein the form-in-place gasket is between about10 micrometers and about 50 micrometers thick.
 7. The apparatus of claim1, wherein the inlets are spaced at uniform intervals.
 8. The apparatusof claim 7, wherein the uniform interval is selected from the groupconsisting of about 4.5 mm, about 9 mm, and about 2.25 mm.
 9. Theapparatus of claim 1, wherein the form-in-place gasket is between about100 micrometers and about 3 millimeters wide.
 10. An apparatuscomprising a plurality of assay chambers, the apparatus comprising, inorder, a substrate, a form-in-place gasket, and a cover, wherein theform-in-place gasket, the substrate, and the cover togethersubstantially define a plurality of assay chambers, wherein theform-in-place gasket forms a permanent seal between the form-in-placegasket and the cover producing a plurality of fluid containmentstructures, each fluid containment structure housing at least one assaychamber, wherein each fluid containment structure is contained on thetop by the cover, wherein each fluid containment structure is containedon the side by the form-in-place gasket, wherein each fluid containmentstructure is contained on the bottom by the substrate, wherein theform-in-place gasket, the substrate, and the cover together furtherdefine a plurality of inlets and outlets, wherein the inlets and outletsare through the form-in-place gasket in a plane parallel with thesubstrate and the cover, wherein the inlet and the outlet are disposedon opposite ends of each assay chamber, wherein each assay chamber is influid communication with an inlet and an outlet, wherein the assaychambers are spaced at uniform intervals, wherein a pair of the assaychambers are in fluidic communication via a channel, wherein the pair ofassay chambers are positioned in series with one another, wherein theassay chambers are in fluid communication with an external fluiddispensing system, wherein the form-in-place gasket is between about 10micrometers and about 250 micrometers thick, wherein the form-in-placegaskets consists of gasket materials selected from at least thefollowing: silicon sealants, urethanes, polysulfides, latex, and acrylicsealants, and wherein the cover and the substrate are made of differentmaterials than the form-in-place gasket.
 11. The apparatus of claim 10,wherein the inlets are fluidically coupled to an external fluiddispensing system that handles multiple fluids in parallel.
 12. Theapparatus of claim 11, wherein the inlets are spaced at uniformintervals.
 13. The apparatus of claim 12, wherein the uniform intervalis selected from the group consisting of about 4.5 mm, about 9 mm, andabout 2.25 mm.
 14. The apparatus of claim 13, further comprising aplurality of analysis components, each analysis component being inoperable association with one or more of the assay chambers.
 15. Theapparatus of claim 14, wherein the analysis components are arrays, andwherein each assay chamber is in operable association with one or moreof the arrays.
 16. The apparatus of claim 13, wherein the form-in-placegasket is between about 10 micrometers and about 100 micrometers thick.17. The apparatus of claim 10, wherein the form-in-place gasket isbetween about 10 micrometers and about 100 micrometers thick.
 18. Theapparatus of claim 10, wherein the form-in-place gasket is between about100 micrometers and about 3 millimeters wide.
 19. An apparatuscomprising a plurality of assay chambers, the apparatus comprising, inorder, a substrate, a form-in-place gasket, and a cover wherein theform-in-place gasket, the substrate, and the cover togethersubstantially define a plurality of assay chambers, wherein theform-in-place gasket forms a permanent seal between the form-in-placegasket and the cover producing a plurality of fluid containmentstructures, each fluid containment structure housing at least one assaychamber, wherein each fluid containment structure is contained on thetop by the cover, wherein each fluid containment structure is containedon the side by the form-in-place gasket, wherein each fluid containmentstructure is contained on the bottom by the substrate, wherein theform-in-place gasket, the substrate, and the cover together furtherdefine a plurality of inlets and outlets, each assay chamber in fluidcommunication with an inlet and an outlet, wherein the inlets andoutlets are through the form-in-place gasket, wherein the inlet and theoutlet are disposed on opposite ends of each assay chamber, wherein theinlets are coupled to an external fluid dispensing system, wherein theinlets are spaced at uniform intervals, wherein the form-in-place gasketis between about 10 micrometers and about 250 micrometers thick, andwherein the form-in-place gasket is formed from gasket materialsselected from at least the following: silicon sealants, urethanes,polysulfides, latex, and acrylic sealants, and wherein the cover and thesubstrate are made of different materials than the form-in-place gasket.20. The apparatus of claim 19, wherein the assay chambers are spaced atuniform intervals.
 21. The apparatus of claim 20, wherein the uniforminterval is selected from the group consisting of about 4.5 mm, about 9mm, and about 2.25 mm.
 22. The apparatus of claim 19, further comprisinga plurality of analysis components, each analysis component being inoperable association with one or more of the assay chambers.
 23. Theapparatus of claim 22, wherein the analysis components are arrays, andwherein each assay chamber is in operable association with one or moreof the arrays.
 24. The apparatus of claim 19, wherein the form-in-placegasket is between about 10 micrometers and about 100 micrometers thick.25. The apparatus or claim 19, wherein the form-in-place gasket isbetween about 10 micrometers and about 50 micrometers thick.
 26. Theapparatus of claim 19, wherein the form-in-place gasket is between about100 micrometers and about 3 millimeters wide.
 27. The apparatus of claim19, wherein a pair of the assay chambers are in fluidic communicationvia a channel, wherein the pair of assay chambers are positioned inseries with one another.